| Canine breast tumor is one of the common diseases in veterinary clinic. The prevention and cure of the disease are closely watched by PET doctors. As there are many similarities between the development of canine mammary tumors and human breast tumors, it can be used as an excellent model to study the development of anticancer drugs. Cisplatin is a first line chemotherapeutic drug for the treatment of many tumors, its broad-spectrum anti-cancer effect has been the people’s praise and clinically used for ovarian cancer, prostate cancer, lung cancer and osteosarcoma strength tumors have a very good effect. While study on the domestic and foreign literature and cisplatin in canine mammary tumor, and the anticancer mechanism of cisplatin induced canine breast cancer has not been reported. This experiment to explore the cisplatin induced canine breast tumor cell apoptosis and its impact on the factor related apoptosis and on the future of canine mammary tumors clinical treatment and scheme formulation is of great significance, but also provide a theoretical basis for study of human breast tumors.The culture of canine breast tumor cell line CHMp(primary tumor cells) and CHMm(metastatic cells) were used as a model in vitro. After treatment with different concentrations of cisplatin 2.5 μmol/L, 5 μmol/L and 10 μmol/L, 12 h, 24 h and 48 h were cultured. Observe and record the cell proliferation and growth morphology by inverted microscope. IC50 value and cytotoxicity of cisplatin on CHMm/CHMp were detected by MTT assay. Also detect the analysis of the effect of apoptosis and cell cycle arrest CHMm/CHMp by flow cytometry. Finally the real-time fluorescent quantitative PCR and Western blot were used to detect the CHMm/CHMp apoptosis related factor Akt, Bcl-2, NFκB gene expression and protein expression. The experimental data was taken into the SPSS19.0 software statistical analysis to get the results of the experiment.Results show:(1)MTT method was used to detect the IC50 values of CHMm and CHMp were 4.86 μmol/L and 5.58 μmol/L, respectively. At the same time, the control group and treated group were very significant difference(P<0.01), and under the same concentration between each experimental group, it is extremely significant difference(P<0.01), with increasing concentrations of cisplatin,prolong action time and inhibition of two cell lines rate gradually increased, there are double the concentration time dependence.(2)The cell wall growth of CHMm and CHMp in the control group was observed under microscope, and the cell body was in a diamond shape, the contour was clear, and the boundary was obvious. Cisplatin at a dose of 5 μmol/L, tumor cells of experimental group was larger thancontrol group in the cell body, the cytoplasm is transparent, adherent ability. when the dose of cisplatin was 10 μmol/L, cells in the experimental group was turned round, cytoplasm turbid,refraction was poor and under high magnification observation can see black particulate matter increased, with prolongation of culture time, the more cells appeared apoptosis phenomenon.(3)According to the flow cytometry was used to detect apoptosis. Each experimental group differences were significant(P<0.05), With increasing concentration of platinum, the CHMm/CHMp cell clusters are on the move, the apoptotic cells gradually increased, inhibition of cell rate increased. Detection of cell cycle showed that CHMm/CHMp increased with the concentration of cisplatin, G1 phase ratio difference was not significant(p>0.05), S phase ratio was significantly increased(P<0.01), G2 ratio gradually decreased(p<0.05). It was showed that cisplatin could inhibit the mitosis of tumor cells, arrest the cell in S phase.(4)Fluorescence quantitative PCR detection of apoptosis gene expression showed that the CHMm/CHMp control group and the experimental group were significantly different(p<0.01),with the same concentration of cisplatin, the time difference between the two groups was significant(p<0.05). Along with the extension of time, the control group of Akt and Nfκb and the expression of bcl-2 gene expression showed an increasing trend, and the experimental group in Suishun platinum concentration increased, Akt and bcl-2 gene expression was significantly decreased, the Nfκb gene expression was markedly increased. The expression levels of AKT, Nfκb and Bcl-2 in CHMm and CHMp were different.(5)The expression of apoptosis factor protein was proved by Blot Western. At the same time point of 24 h, the expression of Akt and bcl-2 protein content decreased with the increase of the concentration of cisplatin, while the expression of Nfκb protein increased. The protein expression of Akt and Bcl-2 decreased in the increasing of the time with the same cisplatin concentration of 5mol/L, and the protein content of Nfκb gradually increased in the increasing of the time. The expression of Akt and bcl-2 protein was down regulated, and the expression of Nfκb protein was up-regulated. |
PDF Full Text Request